Human MICAL1: Activation by the small GTPase Rab8 and small‐angle X‐ray scattering studies on the oligomerization state of MICAL1 and its complex with Rab8

Abstract

Human MICAL1 is a member of a recently discovered family of multidomain proteins that couple a FAD-containing monooxygenase-like domain to typical protein interaction domains. Growing evidence implicates the NADPH oxidase reaction catalyzed by the flavoprotein domain in generation of hydrogen peroxide as a second messenger in an increasing number of cell types and as a specific modulator of actin filaments stability. Several proteins of the Rab families of small GTPases are emerging as regulators of MICAL activity by binding to its C-terminal helical domain presumably shifting the equilibrium from the freeauto-inhibitedconformation to the active one. We here extend the characterization of the MICAL1-Rab8 interaction and show that indeed Rab8, in the active GTP-bound state, stabilizes the active MICAL1 conformation causing a specific four-fold increase of k cat of the NADPH oxidase reaction. Kinetic data and small-angle X-ray scattering (SAXS) measurements support the

GRAF2, WDR44, and MICAL1 mediate Rab8/10/11–dependent export of E-cadherin, MMP14, and CFTR ΔF508

“…Cargo selection proceeds by an unknown mechanism, which may be related to a specific lipid environment such as an enrichment in PI(4,5)P 2 or cholesterol (Ling et al, 2007;Tan et al, 2015;Annabi et al, 2001;Seveau et al, 2004), partitioning in highly curved membranes, direct binding of Rabs or adapters (Ling et al, 2007;Lock and Stow, 2005;Lau and Mruk, 2003), and/or cytoskeletal retention (MacDonald et al, 2018). (B) GTP-bound Rab10 and Rab8a/b recruit and activate MICAL1, which may bind two Rabs simultaneously (Rai et al, 2016;Esposito et al, 2018). MICAL1 may also bind directly to the membrane via its PI(4,5)P 2 -binding CH domain (Alqassim et al, 2016).…”

MICAL1 activation by PAK1 mediates actin filament disassembly

“…When co-expressed with CDC42 G12V and PAK1, only the N1, N2, N3 and C3 constructs that all contained the MO and/or CH domains were co-immunoprecipitated ( Figure 1F ). Thus, unlike RAB GTPases or Plexin that bind to the carboxyl-terminal domain [10, 13, 14], PAK1 binds to the same MO + CH domains as F-actin [8, 27].…”

“…Structural analysis shows that the majority of MICAL1 exists in a closed inactive conformation autoinhibited by the carboxyl-terminal region, which can be relieved by the binding of proteins such as RAB GTPases to the CC/RBD region to open and activate MICAL1 [8, 10]. The affinity of the CC/RBD for RAB proteins was found to be dependent on the positions of the first and second α-helices [10], raising the possibility that PAK1 phosphorylation of S817, located between the LIM and CC/RBD protein binding domains, and S960, located at the end of α-helix1 that leads to a turn that couples to the start of α-helix2 ( Figure 3E ), might affect protein binding. To identify MICAL1 interacting proteins affected by PAK1 phosphorylation, FLAG-MICAL1 co-expressed with active CDC42 and PAK1 KD or PAK1 WT was immunoprecipitated, followed by tryptic digestion, quantification and MS/MS analysis of co-precipitated proteins.…”

“…Of particular interest was the identification of RAB10 as being preferentially associated with phosphorylated MICAL1 ( Figures 4A and 4C ). Although several lines of evidence have shown that MICAL1 associates with RAB GTPases in vitro [10], in cells [12] and in yeast two-hybrid screens [9], there has been no indication that MICAL1 post-translational modifications influence RAB GTPase binding. To determine whether MICAL1 phosphorylation affected RAB10 binding, FLAG-MICAL1 co-expressed with CDC42 and PAK1 WT or PAK1 KD was also expressed with enhanced green fluorescent protein (eGFP) or eGFP-RAB10 ( Figure 4D ).…”

“…Immunoprecipitation of FLAG-MICAL1 resulted in the specific coimmunoprecipitation of eGFP-RAB10, which was significantly greater when S960 was phosphorylated by PAK1 ( Figure 4D , right panel; Figure 4E ). These results reveal for the first time that there is a two-way relationship between MICAL1 and RAB GTPase binding; in addition to RAB GTPase binding inducing conformational changes that increase activity [10, 12], MICAL1 phosphorylation by PAK1 increases RAB GTPase binding.…”